RNA interference (RNAi) is an evolutionally conserved mechanism of gene regulation by which small double-stranded RNA (dsRNA) molecules inhibit translation or degrade complementary mRNA sequences (Elbashir, S. M., et al. (2001) Nature 411, 494-8; Fire, A., et al. (1998) Nature 391, 806-11; Zeng, Y., et al., (2003) Proc Natl Acad Sci USA 100, 9779-84.). Synthetic dsRNA duplexes, termed small interfering RNAs (siRNAs), mimic naturally processed dsRNAs [e.g. microRNAs (miRNAs)] to exploit endogenous enzymatic machinery and enter the RNAi pathway.
More recently, short dsRNAs have also been shown to induce gene expression in a phenomenon referred to as RNA activation (RNAa) (Janowski, B. A., et al. (2007) Nat Chem Biol 3, 166-73; Li, L. C., Okino, S. T., et al. (2006) Proc Natl Acad Sci USA 103, 17337-42; Place, R. F. et al. (2008) Proc Natl Acad Sci USA 105, 1608-13.). This class of dsRNA molecule—termed small activating RNAs (saRNAs) to distinguish from siRNAs—triggers an effect opposite to that of RNAi. Several models of RNAa have been described including transcriptional activation by targeting promoter-derived sequences (Li, L. C., et al. (2006) Proc Natl Acad Sci USA 103, 17337-42; Place, R. F., et al. (2008) Proc Natl Acad Sci USA 105, 1608-13; Kuwabara, T., et al. (2004) Cell 116, 779-93; Wang, X., et al. (2008) Nature 454, 126-30.) and/or gene antisense transcripts (Morris, K. V., et al. (2008) PLoS Genet 4, e1000258; Schwartz, J. C., et al. (2008) Nat Struct Mol Biol 15, 842-8.). Regardless of the target, it has become clear that RNAa has potential to induce the expression of a variety of genes. As such, RNAa offers a promising new therapeutic strategy for diseases that can be corrected by stimulating gene expression.
Identifying features of the RNAi pathway have improved its therapeutic application and development as a laboratory tool. Early studies evaluating the rate of RNAi activity defined the optimal window for target knockdown and functional gene analysis (Takahashi, Y., et al. (2006) Biotechnol Bioeng 93, 816-9; Harborth, J., et al. (2001) J Cell Sci 114, 4557-65.). Medicinal chemistry also enabled usage and identification of modifications to siRNAs that improved mechanistic analysis and pharmacological properties (Manoharan, M. (2004) Curr Opin Chem Biol 8, 570-9; de Fougerolles, A., et al. (2005) Methods Enzymol 392, 278-96; Layzer, J. M., et al. (2004) Rna 10, 766-71). Understanding the functional nuances of RNAa, and applying this understanding to the development of improved saRNAs is of equal importance. This application addresses these issues.